PROJECT SUMMARY Accurate transmission of genetic information is required for growth, proliferation and development of tissues and organisms. Faithful segregation of chromosomes involves many events, including the duplication of microtubule organizing centers (MTOCs), known as centrosomes in metazoans and spindle pole bodies (SPBs) in fungi, once and only once per cell cycle. In order for the cytoplasmic microtubule apparatus emanating from the MTOCs to access the chromosomes in the nucleus, the nuclear envelope (NE) must also be remodeled during cell division. In some cell types the NE entirely or partially disassembles while in others it remains intact. Centrosomes and SPBs adapt differently to these two mechanisms, as illustrated by the NE insertion of SPBs in both Saccharomyces cerevisiae and Schizosacchromyces pombe. Although centrosomes and SPBs are structurally distinct, both types of MTOCs duplicate, grow and interact with the NE. Defects in any of these events could lead to spindle errors, which often result in the gain or loss of a chromosome (aneuploidy). Aneuploidy in yeast often can be tolerated, but in humans it is frequently associated with cancer due to changes in uncovered recessive mutations or alterations in protein complexes. This proposal seeks to elucidate conserved principles used by the cell to restrict centrosome/SPB duplication to once per cell cycle, to ensure the MTOC reaches a size where nucleation capacity is sufficient for chromosome segregation and to insert or tether centrosomes/SPBs to the NE. Our innovative two-color structured illumination microscopy (SIM) with single-particle averaging (SPA) approach sets our work apart because we are able to resolve SPB features and duplication intermediates required for these events that were not observed using electron microscopy, biochemical, genetic or other super-resolution methods. In this work, we build upon the observations we have made and further extend imaging technology by pairing fluorescence resonance energy transfer (FRET) with SIM. This advancement allows us to study protein-protein interactions during SPB duplication and compare them to protein-protein interactions in a mature SPB to understand how centrosome formation is controlled. Our preliminary data suggests that physical interactions are dynamic and change throughout the SPB duplication cycle, an idea that we will further investigate by examining phosphorylation and other cell cycle-dependent modifications. Using SIM, we can visualize the SPB pore?the ring-like structure that anchors the soluble SPB in the NE. The mechanisms by which this structure and the related pore at nuclear pore complexes form is poorly understood. Because we can observe the SPB pore in yeast, we can dissect the molecular events used by cells to create this hole in the NE. Our overall objective is to determine mechanisms that coordinate centrosome duplication with DNA replication (Aim1), elucidate the molecular events that allow the SPB to insert into the NE (Aim 2) and study how SPB size and microtubule nucleation is controlled (Aim 3) using imaging in combination with genetic and molecular methods.